PROJECT SUMMARY: This supplement will critically update the instrumentation available for all aspects of the parent grant, Riboswitch mechanism unraveled at the single molecule level, as well as all 3.5 NIGMS R01 grants of the PI that are currently pending conversion into a single R35 MIRA award, entitled The RNA nanomachines of gene expression dissected at the single molecule level. The most critical aspects of the proposed instrumentation, the ONI Nanoimager S, are its versatility, turnkey readiness, and ease of use. These features will dramatically facilitate access by the diverse group of postdoctoral fellows, graduate students and undergraduate students in the PI?s group to a plethora of single molecule fluorescence microscopy tools. In turn, these tools are leveraged directly by the parent grant, which is focused on dissecting the mechanisms of the nanoscale RNA machines of gene expression at the single molecule level. Building on our group?s 20-year expertise in this space, we aim to: 1.) Apply our established mechanistic enzymology approaches to an ever broader set of RNAs involved in regulating transcription, translation and splicing, seizing the opportunities arising from the continuing discoveries of new functional RNAs. 2.) Push the limits of our approaches to be able to probe increasingly complex biological contexts and mechanisms since unexpected discoveries often await where individual RNA nanomachines interact. In pursuit of these aims, we will address the unifying hypothesis that dynamic RNA structures are a major determinant of the outcomes of gene expression, as exemplified by the fact that nascent RNA structure has a significant impact on both transcription and translation in the form of regulatory riboswitches embedded near the 5? ends of bacterial mRNAs. Exemplifying the power of our scientific approach to address our hypothesis, we recently combined single-molecule, biochemical and computational simulation tools to show that transcriptional pausing at a site immediately downstream of a riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced consensus pause sequence, and electrostatic and steric interactions with the exit channel of bacterial RNA polymerase. We posit that many more examples of similarly intimate structural and kinetic coupling between RNA folding and gene expression remain to be discovered, leading to the exquisite regulatory control enabling all life processes. To reveal more such couplings, we will probe the dynamics of additional gene expression complexes using a tailored combination of single molecule fluorescence resonance energy transfer (smFRET) and Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS). A major bottleneck in these pursuits so far has been the steep learning curve associated with our two home-built microscopes that keeps new group members from making significant contributions until they have completed 1- 2 years of training. We anticipate that addition of the ONI Nanoimager S to our microscopy arsenal will transform the speed of our progress by introducing an easy-to-use instrument that beginning postdocs, graduate and undergraduate students can quickly use independently until they ?graduate? to the home-built microscopes.